4. Discussion
Although a bioreactor with reciprocal mixing cannot be simply compared
to conventional bioreactors with rotary paddles, the cultivation
conditions of CHO cells in both bioreactors, in terms of cell growth,
damage, and lactate production were compared in this report. There was
no significant difference between the bioreactors in terms of cell
proliferation and density during exponential phase. However, in the
reciprocal bioreactor, the transition from stationary to death phase was
slower, and high cell viability was maintained for a longer period than
in the bioreactor with rotary paddles. Moreover, lactate dehydrogenase
(LDH) activity in the medium of reciprocal bioreactor was much lower
than in the rotary bioreactor. Since LDH is a cell damage marker, shear
stress on cells in the reciprocal bioreactor was shown to be overall
lower than in the rotary bioreactor. The condition of cells, after
entering early stationary phase, is important for industrial production
of targeted proteins. Therefore, maintaining high cell viability after
stationary phase is critical for high-quality production.
When shear stress in bioreactors was analyzed by CFD, strong shear
stress generated by separation vortex was observed around the rotary
paddles, and cells were found to be exposed to it throughout the
cultivation process. On the other hand, maximum shear stress in
reciprocal mixing was observed around the plates when they turned at the
top and bottom dead points. Shear stress in reciprocal mixing was
minimized when the plates passed through the middle point of stroke.
This non-steady-state mixing of reciprocal motion could dramatically
reduce the accumulation of cell damage, thereby affecting the
physiological conditions of living cells.
Influence of shear stress on cell damage has been investigated not only
in bioreactors, but also in microfluid devices and blood vessels
(Mitchell, 2013; Odeleye, 2014; Model, 2014). Although many researchers
have tried to clarify how shear stress affects gene expression and
physiological conditions (Nurhayati, 2018; Jayagopal, 2019), the kind of
cellular systems that sense the effect of shear stress on gene
expression still remains unknown. Even if a type of shear stress induced
gene expression in one cell type, it may not affect the same in other
cell lines (Akimoto, 2000; Novak, 2019). Therefore, how reciprocal
mixing could influence gene expression and intracellular physiological
conditions, resulting in the maintenance of high cell viability and
target protein expression, needs to be clarified in future.
Recently, Eto’s group reported a bioreactor with reciprocal mixing to be
effective for platelet generation from human iPS-derived megakaryocytes
(Ito, 2018). Platelet biogenesis from megakaryocytes requires blood
flow-dependent shear stress (Junt, 2008). While other bioreactors with
rotary paddles or culture bags could not produce sufficient and
appropriate levels of shear stress, a bioreactor with reciprocal motion
could generate proper turbulent energy in the culture medium, thereby
inducing in-vitro thrombopoiesis.
Development of a bioreactor, till date, has mainly focused on the growth
of cultured organism, production of targeted compounds or proteins, and
homogeneity of culture broth. A wide variety of ideas, including
different kinds of mixing impellers, and rotating or see-sawing culture
vessels have been developed over the years (Birch, 1990; Rotenberg,
2012). However, mixing and dispersing actions are inversely proportional
to shear stress, in case of conventional bioreactors, making it
difficult to optimize the culture conditions (Wyma, 2018). In this
study, we have introduced a novel concept of bioreactors for culturing
animal cells, and clarified the specific characteristics of a bioreactor
with reciprocal mixing in comparison to those of a conventional
bioreactor with rotary paddles. Although the reason behind the
appreciable effect of reciprocal mixing on cell growth remains unknown,
shear stress generated by reciprocal motion might be physiologically
acceptable for cell growth. Studies are ongoing for understanding the
processes occurring in cells when they are exposed to reciprocal shear
stress.